The goal of this grant application is to develop a simpler and more effective approach to generate dendritic cells (DC) for cancer immunotherapy. A key and limiting step in the process of generating human monocyte derived DC for clinical applications - currently the most widely used DC generation protocol - is the maturation step. The proposal of this grant application, supported by preliminary data, is that the ex vivo DC maturation step can be eliminated and replaced by injecting the immature DC into "inflamed" (or "primed") tissue, namely tissue that was exposed to agents that induce a microenvironment conducive to DC maturation and migration. The central hypothesis of this grant application is that by using appropriate agents it will be possible to recapitulate the physiological condition occurring during pathogen infection in a manner that will lead to the optimal conditions for DC maturation, migration and function. The specific objectives of the proposed studies are to identify the appropriate (combination of) agents to induce the best possible outcome, namely the generation of a potent antitumor immune response which is equal or superior to using ex vivo matured DC. Murine studies will explore the optimal conditions for in situ maturation using classical adjuvants and whether local injection of biological response modifiers (BRMs) in the form of mRNA can contribute to the generation of potent immunostimulatory DC. The ability to migrate to lymph nodes and immunostimulatory capacity of the injected DC will be used to monitor the success of the protocol. Human studies will explore whether in situ maturation can enhance the lymph node migration of the human monocyte-derived DC in cancer patients. The in situ maturation approach offers several potential advantages over current DC generation protocols: a) It recapitulates the physiological conditions for DC maturation and hence would result in a more potent immune response, b) In situ maturation eliminates an in vitro culture step which is the setting of this patient-specific cell therapy protocol represents a considerable simplification, and c) In situ maturation obviates the dependence on expensive biological reagents used for ex vivo DC maturation which are replaced by readily available and inexpensive reagents. The studies described in this application will set the stage for clinical trials using immunological end points to determine whether "in situ maturation" generates potent APC to stimulate T cell immunity in cancer patients.